In total internal reflection microscopy, the refractive behavior of light as it makes the transition from an optically denser medium to an optically thinner medium is utilized. Thus, for example, the transition from cover glass (n1=1.518) to water (n2=1.33) yields a critical angle of 61°, the angle of total reflection. Under the conditions of total reflection (angle ≧61°), a standing evanescent wave is formed in the medium with the lower refractive index. The intensity of this wave drops exponentially relative to the distance from the interface. For this reason, fluorophores located far away from the interface are not excited. The background fluorescence is considerably reduced. The image contrast is improved in this process and the resolution is markedly raised at the same time. A prerequisite for the utilization of the phenomenon described above is a sufficiently large difference between the refractive indices of the cover glass and of the medium.
US 2002/0097489 A1 describes a microscope with evanescent illumination of a specimen. The microscope comprises a white-light source whose light is coupled into the specimen slide via a slit aperture through the microscope objective for purposes of evanescent illumination. The illumination light propagates in the specimen slide due to total internal reflection, whereby the specimen is only illuminated in the region of the evanescent field that extends from the specimen slide. Microscopes of this type are known by the acronym TIRFM (Total Internal Reflection Fluorescent Microscope). The z-resolution of TIRF microscopes is exceptionally good owing to the fact that the evanescent field extends only about 100 nm into the specimen.
DE 101 08 796 A1 describes a high-aperture objective, particularly for TIRF applications. The objective consists of a first lens having a positive refractive power, a second lens having a negative refractive power, whereby the focal distance ratio between the two lenses lies within the range from −0.4 to −0.1 and the total refractive power is greater than zero. Moreover, the objective comprises two positive lenses whose ratio of the diameter to the focal distance is greater than 0.3 and smaller than 0.6. Furthermore, the objective comprises a negative lens and a collector lens, whereby the negative lens faces the front group and the focal distance ratio between the negative lens and the collector lens is between −0.5 and −2.
DE 102 17 098 A1 describes an incident-illumination array for TIRF microscopy. The incident-illumination array contains a source of illumination that, during operation, emits a polarized illuminating beam bundle that propagates at an angle relative to the optical axis, and the array also comprises a deflecting device that deflects the illuminating beam bundle and couples it into the objective parallel to the optical axis. With this incident-illumination array, it is provided that the illuminating beam bundle emitted by the source of illumination has s-polarization and p-polarization directions with a phase differential and the deflecting device reflects the illuminating beam bundle x times, wherein x=(n×180−d)/60°.
DE 101 43 481 A1 describes a microscope for TIRM (Total Internal Reflection Fluorescent Microscopy). The microscope has a housing and an objective. The illumination light emitted by an illumination device can be coupled in by means of an adapter that can be slid into the microscope housing.
US 2004/0001253 A1 describes a microscope with an optical illumination system that allows a simple switching over between evanescent illumination and reflection illumination. The illumination system comprises a source of laser light whose light is coupled into an optical fiber. Moreover, a coupling-out optical system is provided that focuses the light coming out of the fiber in a rear focal point of the microscope objective. The optical fiber can be slid in a plane perpendicular to the optical axis of the microscope objective.
DE 102 29 935 A1 describes a device for the coupling in of light in a microscope. This is where a light-conducting fiber configured as a slider in the light field aperture plane directs the laser light onto the preparation. The present invention is particularly well-suited for the TIRF method.
In scanning microscopy, a specimen is illuminated with a light beam so that the detection light emitted by the specimen can be observed as reflection light or fluorescent light. The focus of an illumination light beam bundle is moved in a specimen plane by means of a controllable beam-deflecting device, usually by tilting two mirrors, whereby the deflection axes are usually perpendicular to each other, so that one mirror deflects in the x-direction while the other one deflects in the y-direction. The mirrors are tilted by means of, for instance, galvanometer setting elements. The output of the detection light coming from the object is measured as a function of the position of the scanning beam. Normally, the setting elements are fitted with sensors in order to determine the current position of the mirror. Especially in the case of confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a source of light, a focusing optical system, with which the light from the source is focused onto a pinhole (the so-called excitation aperture), a beam splitter, a beam-deflecting device for beam control, a microscope optical system, a detection aperture and the detectors to detect the detection light or fluorescent light. The illumination light is coupled in by means of a beam splitter. The fluorescent light or reflection light coming from the object returns to the beam splitter via the beam-deflecting device, passes through the beam splitter in order to be subsequently focused on the detection aperture downstream from which the detectors are located. This detection arrangement is called a descan arrangement. Detection light that does not stem directly from the focus region takes a different light path and does not pass through the detection aperture, so that point information is obtained that yields a three-dimensional image as a result of sequential scanning of the object with the focus of the illumination light beam bundle. For the most part, a three-dimensional image is obtained by means of layerwise image data acquisition.
With the microscopes known from the state of the art, the evanescent illumination is regularly coupled in within the scope of two-dimensional solutions, even if the adjustment unit used in such cases is always configured one-dimensionally. Thus, the coupling-in is done, for instance, by means of a so-called neutral splitter, that is to say, by means of a mirror that reflects light to a certain extent and otherwise transmits light. Coupling-in by means of a dichroitic splitter is also known. In this case, it is a special mirror that, except for one specific wavelength, reflects all other wavelengths. Another known approach is coupling-in by means of a polarization splitter. Here, the lasers for the evanescent illumination (TIRF illumination) and the laser for the conventional epi-fluorescent illumination are polarized orthogonally with respect to each other and then combined. As a one-dimensional possibility for coupling in the requisite source of radiation, it is likewise already known to use small additional mirrors in the illuminating beam path for the epi-fluorescent illumination.
When it comes to realizing evanescent illumination (TIRF illumination), the state of the art fundamentally couples in the illumination light either on the objective side or on the condenser side. The back-reflections created by the evanescent illumination are, in turn, coupled out and, normally, reflected into a light trap in order to avoid scattered light or parasitic reflections. So far, the returning reflection light is actually detrimental some and consequently has to be “disposed of”.
DE 103 09 269 A1 describes the approach of utilizing the illumination light for purposes of laser protection. Towards this end, when the illumination light is coupled in, part of the light output is coupled out and subsequently mirrored onto a detector. By the same token, part of the light output is mirrored out within the scope of the coupling-out and, in turn, focused onto a detector. If the intensity ratio falls below a certain value, then the light source of the coupled-in illumination light—usually the illumination light of a coupled-in laser—is switched off by means of a protective device provided especially for this purpose.
The microscopes and methods known for total internal reflection microscopy from the state of the art are disadvantageous insofar as, when the beam is directed rotation-symmetrically, it is not possible to quantitatively determine the penetration depth that is to be achieved for the evanescent field that is being created in the specimen. This is due to the normally unknown refractive index of the specimen to be examined. If the refractive index of the specimen is known, the penetration depth can be calculated in a familiar manner from the known refractive index and angle of incidence of the evanescent illumination light beam relative to the specimen. Moreover, when different specimens are being examined, one is confronted with different refractive indices so that it is difficult to calculate the angle of total reflection. Consequently, an automatic setting of the evanescent illumination seems to be ruled out. When the beam is directed non-rotation-symmetrically, a homogeneously rotation-symmetrical irradiation of the TIRF illumination is hardly possible.